A fundamental gap exists in our understanding of the signals required for progenitor cells to differentiate into tendon and ligament fibroblasts. Strategies for directing precursor cell differentiation into mature ligament fibroblasts are a critical, unmet need in the development of functional ligament tissues. Providing a signaling environment capable of directing progenitor cell differentiation along a ligament lineage is a major challenge facing the field of ligament engineering. The long term goal is to further understand normal ligament biology as a means of discovering novel ways to improve the treatment of ligament injuries. The objective of this application is to develop a strategy that enhances mesenchymal stem cell differentiation along a ligament lineage. Critical signals can be intracellular (transcription factors and signaling proteins) or environmental (mechanical strain and three- dimensional environment surrounding the cells). The central hypothesis is that overexpression of the transcription factor scleraxis will provide the appropriate signaling environment for mesenchymal stem cells to progress into mature ligament fibroblasts useful for ligament engineering. Thus, the proposed research is relevant to the NIH's mission to foster innovative research strategies to advance the nation's capacity to improve health. The hypothesis will be tested by pursuing two specific aims: (1) Identify a mechanism by which stem cells grown in vitro will be induced to differentiate along a ligament lineage, and (2) Establish conditions of mechanical strain that will enhance differentiation of scleraxis-modified stem cells. In the first aim, a mouse mesenchymal stem cell line will be engineered to overexpress scleraxis and cells will be cultured in three-dimensional collagen gels under conditions of static strain (tension). Cell differentiation will be determined via gene expression and protein production for markers of a ligament phenotype. In the second aim, mesenchymal stem cells overexpressing scleraxis will be cultured in three-dimensional collagen gels under variable conditions of cyclic mechanical strain. Induction of a ligament phenotype (cell morphology, gene expression, and protein production) and cell vitality will be assessed. This innovative research focuses on utilizing a novel transcription factor to solve one of the most challenging aspects of ligament engineering: production of a function-appropriate extracellular matrix composite. The proposed research is significant, because it is expected to improve understanding of how stem cells are driven along a ligament lineage, thus advancing the fields of ligament engineering and regeneration. PUBLIC HEALTH RELEVANCE: The proposed studies focus on an important area of cell differentiation for tissue regeneration. This information combined with existing tissue engineering technologies will significantly advance the field of functional ligament engineering. The proposed research has public health relevance, because understanding the key to cell differentiation into ligament fibroblasts will improve tissue healing and ultimately the quality of life for people suffering from ligament injuries.